A New Series of Indeno[1,2-c]pyrazoles as EGFR TK Inhibitors for NSCLC Therapy

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death throughout the world. Due to the shortcomings of traditional chemotherapy, targeted therapies have come into prominence for the management of NSCLC. In particular, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapy has emerged as a first-line therapy for NSCLC patients with EGFR-activating mutations. In this context, new indenopyrazoles, which were prepared by an efficient microwave-assisted method, were subjected to in silico and in vitro assays to evaluate their potency as EGFR TK-targeted anti-NSCLC agents. Compound 4 was the most promising antitumor agent towards A549 human lung adenocarcinoma cells, with an IC50 value of 6.13 µM compared to erlotinib (IC50 = 19.67 µM). Based on its low cytotoxicity to peripheral blood mononuclear cells (PBMCs), it can be concluded that compound 4 exerts selective antitumor action. This compound also inhibited EGFR TK with an IC50 value of 17.58 µM compared to erlotinib (IC50 = 0.04 µM) and induced apoptosis (56.30%). Taking into account in silico and in vitro data, compound 4 stands out as a potential EGFR TKI for the treatment of NSCLC.


Introduction
Non-small cell lung cancer (NSCLC), which accounts for 85% of all lung cancers, represents the most common cause of cancer-related mortality worldwide [1,2], with a 5-year survival rate of less than 15% [3]. The best therapy option for NSCLC patients continues to be the complete surgical resection of the tumor [4]. However, most NSCLC patients are diagnosed at advanced or metastatic stages (stage III/IV), when surgery is no longer an option. In these cases, radiotherapy and chemotherapy are important therapeutic approaches for unresectable NSCLC [4][5][6][7].
Platinum-based chemotherapy is an existing Food and Drug Administration (FDA)approved strategy in the management of several common cancer types, including NSCLC, and has many benefits in some cases [8,9]. Traditional chemotherapeutic agents eliminate not only rapidly dividing cancer cells but also normal cells, and therefore these drugs are highly toxic to all cells in the body [8][9][10]. Traditional cytotoxic chemotherapy also causes changes in the normal function of the cells, and correspondingly various side effects such as fatigue, anemia, alopecia, and gastrointestinal complications [8][9][10].
The Infrared (IR), 1 H Nuclear Magnetic Resonance (NMR), 13 C NMR, and High-Resolution Mass Spectrometry (HRMS) data were in agreement with the proposed structures of compounds 1-7. In the IR spectra of compounds 1-7, the absence of a C=O stretching band at 1684 cm −1 [31] confirmed that the ring closure reaction leading to the formation of the dihydroindenopyrazole scaffold occurred efficiently. The C=N and C=C stretching bands were located in the region of 1630-1450 cm −1 . In the 1 H NMR spectra of the compounds, the indene C 4 -CH 2 protons were observed as a singlet at 3.32-3.75 ppm. In the 1 H NMR spectra of compounds 1-7, the protons attached to the C 3 , C 4 , and C 5 carbons of the piperidine ring gave rise to a broad singlet at 1.57 or 1.65 ppm. The signals due to the protons attached to the C 2 and C 6 carbons of the piperidine ring appeared at 3.17-3.23 ppm as a broad singlet. All the other aliphatic and aromatic protons were observed as expected, e.g., Ar-OCH 3 protons as a singlet at 3.93-4.01 ppm, and Ar-CH 3 protons as a singlet at 2.39-3.10 ppm. The dihydroindenopyrazole C 4 -CH 2 and OCH 3 signals were observed at 27.63-30.10 and 52. .69 ppm, respectively. The C 4 , C 3 , and C 5 , C 2 , and C 6 carbons of the piperidine ring gave rise to the peaks at 22 in the 13 C NMR spectra of compounds 1-7. The formation of the dihydroindenopyrazole scaffold was also verified by the HRMS analysis of compounds 1-7. The Infrared (IR), 1 H Nuclear Magnetic Resonance (NMR), 13 C NMR, and High-Resolution Mass Spectrometry (HRMS) data were in agreement with the proposed structures of compounds 1-7. In the IR spectra of compounds 1-7, the absence of a C=O stretching band at 1684 cm −1 [31] confirmed that the ring closure reaction leading to the formation of the dihydroindenopyrazole scaffold occurred efficiently. The C=N and C=C stretching bands were located in the region of 1630-1450 cm −1 . In the 1 H NMR spectra of the compounds, the indene C4-CH2 protons were observed as a singlet at 3.32-3.75 ppm. In the 1 H NMR spectra of compounds 1-7, the protons attached to the C3, C4, and C5 carbons of the piperidine ring gave rise to a broad singlet at 1.57 or 1.65 ppm. The signals due to the protons attached to the C2 and C6 carbons of the piperidine ring appeared at 3.17-3.23 ppm as a broad singlet. All the other aliphatic and aromatic protons were observed as expected, e.g., Ar-OCH3 protons as a singlet at 3.93-4.01 ppm, and Ar-CH3 protons as a singlet at 2.39-3.10 ppm. The dihydroindenopyrazole C4-CH2 and OCH3 signals were observed at 27.63-30.10 and 52.33-59.69 ppm, respectively. The C4, C3, and C5, C2, and C6 carbons of the piperidine ring gave rise to the peaks at 22.68-25.53, 25.39-27.63, and 45.09-52.49 ppm in the 13 C NMR spectra of compounds 1-7. The formation of the dihydroindenopyrazole scaffold was also verified by the HRMS analysis of compounds 1-7.

In Vitro Assays
Compounds 1-7 and erlotinib (positive control) were tested for their cytotoxic effects on A549 human lung adenocarcinoma cells using the MTT test, a tetrazolium-based cell viability assay. Compounds 1, 4, and 7 were found to be more effective than erlotinib. Compounds 1, 4, and 7 exerted their antitumor action towards A549 cells with IC50 values of 7.99, 6.13, and 8.67 µM, respectively, as compared to erlotinib (IC50 = 19.67 µM), an EGFR TKI currently used for NSCLC therapy (Table 1). Other compounds were found to be devoid of cytotoxic activity towards A549 cells (IC50 > 10 µM).
Among halogen substituents (F, Cl, Br), it can be concluded that the p-bromo group significantly enhances anti-NSCLC activity. p-Methyl substitution (-σ and +π effect) also led to marked anti-NSCLC activity, whereas p-methylsulfonyl and p-cyano substitutions (+σ and -π effect) resulted in the loss of anti-NSCLC activity. Scheme 1. The synthetic route for the preparation of compounds 1-7.

In Vitro Assays
Compounds 1-7 and erlotinib (positive control) were tested for their cytotoxic effects on A549 human lung adenocarcinoma cells using the MTT test, a tetrazolium-based cell viability assay. Compounds 1, 4, and 7 were found to be more effective than erlotinib. Compounds 1, 4, and 7 exerted their antitumor action towards A549 cells with IC 50 values of 7.99, 6.13, and 8.67 µM, respectively, as compared to erlotinib (IC 50 = 19.67 µM), an EGFR TKI currently used for NSCLC therapy (Table 1). Other compounds were found to be devoid of cytotoxic activity towards A549 cells (IC 50 > 10 µM). Among halogen substituents (F, Cl, Br), it can be concluded that the p-bromo group significantly enhances anti-NSCLC activity. p-Methyl substitution (−σ and +π effect) also led to marked anti-NSCLC activity, whereas p-methylsulfonyl and p-cyano substitutions (+σ and −π effect) resulted in the loss of anti-NSCLC activity.
Compounds 1, 4, and 7, the most potent anti-NSCLC agents in this series, were investigated for their cytotoxic effects on K562 chronic myelogenous leukemia (CML) cells and peripheral blood mononuclear cells (PBMCs) to determine the selectivity of their antitumor action. Compounds 1, 4, and 7 showed marked anti-CML activity with IC 50  (Table 1). On the other hand, compounds 1, 4, and 7 exerted no significant cytotoxicity towards PBMCs (normal cells). The selectivity index (SI) values of compounds 1, 4, and 7 (27.43, 7.38, and >36.23, respectively) were found to be higher than that of erlotinib (1.50), indicating that their anticancer effects were selective.
Compounds 1, 4, and 7 were subjected to an in vitro mechanistic assay to determine whether their anti-NSCLC activities are related to EGFR TK inhibition or not. None of them inhibited EGFR TK as much as erlotinib (IC 50 = 0.04 ± 0.01 µM). The most potent EGFR TKI in this series was found as compound 4 (IC 50 = 17.58 ± 3.15 µM), followed by compounds 7 (IC 50 = 46.66 ± 7.04 µM) and 1 (IC 50 = 55.29 ± 8.68 µM) (Figure 1a). In particular, p-bromo substituent significantly enhanced EGFR TK inhibitory activity. According to Figure 1b, it can be concluded that compound 4 shows dose-dependent EGFR TK inhibitory potency. Compounds 1, 4, and 7 were subjected to an in vitro mechanistic assay to determine whether their anti-NSCLC activities are related to EGFR TK inhibition or not. None of them inhibited EGFR TK as much as erlotinib (IC50 = 0.04 ± 0.01 µM). The most potent EGFR TKI in this series was found as compound 4 (IC50 = 17.58 ± 3.15 µM), followed by compounds 7 (IC50 = 46.66 ± 7.04 µM) and 1 (IC50 = 55.29 ± 8.68 µM) (Figure 1a). In particular, p-bromo substituent significantly enhanced EGFR TK inhibitory activity. According to Figure 1b, it can be concluded that compound 4 shows dose-dependent EGFR TK inhibitory potency. Kinase selectivity profiling systems, TK-1 (EGFR, HER2, HER4, IGF1R, InsR, KDR, PDGFR-α, and PDGFR-β) and TK-2 (ABL1, BRK, BTK, CSK, FYN A, LCK, LYN B, and SRC), were used to identify the kinase selectivity profile of compound 4. Compound 4 inhibited HER2 and LCK more significantly than erlotinib at 30 µM concentration ( Figure 2). However, its inhibitory effects on HER2 and LCK were not as pronounced as its EGFR inhibitory activity. It is also important to note that the kinase selectivity profile of compound 4 is different from that of erlotinib.
(b) Figure 1. The inhibitory activity of the compounds on EGFR TK. (a) EGFR TK inhibition caused by compounds 1, 4, 7, and erlotinib at 30 µM concentration. (b) The EGFR TK inhibition of compound 4 and erlotinib at different concentrations. All descriptive data were expressed as the Mean ± Standard Deviation (SD). All experiments were repeated three times.
Kinase selectivity profiling systems, TK-1 (EGFR, HER2, HER4, IGF1R, InsR, KDR, PDGFR-α, and PDGFR-β) and TK-2 (ABL1, BRK, BTK, CSK, FYN A, LCK, LYN B, and SRC), were used to identify the kinase selectivity profile of compound 4. Compound 4 inhibited HER2 and LCK more significantly than erlotinib at 30 µM concentration ( Figure  2). However, its inhibitory effects on HER2 and LCK were not as pronounced as its EGFR inhibitory activity. It is also important to note that the kinase selectivity profile of compound 4 is different from that of erlotinib. In general, tumor cells lose their ability to undergo apoptosis, resulting in uncontrolled proliferation, and many antitumor agents trigger the induction of apoptosis to eliminate tumor cells [32]. Moreover, preclinical and clinical studies reveal that inhibition of EGFR, together with enhanced induction of apoptosis, may counter resistance to chemotherapy and radiotherapy [33]. In the current work, the pronounced EGFR TK-targeted anti-NSCLC activity of compound 4 prompted us to evaluate its apoptotic/necrotic effects using the annexin V/ethidium homodimer III staining assay. The results indicated that compound 4 induced apoptosis more than erlotinib, as shown in Figure 3. The percentages In general, tumor cells lose their ability to undergo apoptosis, resulting in uncontrolled proliferation, and many antitumor agents trigger the induction of apoptosis to eliminate tumor cells [32]. Moreover, preclinical and clinical studies reveal that inhibition of EGFR, together with enhanced induction of apoptosis, may counter resistance to chemotherapy and radiotherapy [33]. In the current work, the pronounced EGFR TK-targeted anti-NSCLC activity of compound 4 prompted us to evaluate its apoptotic/necrotic effects using the annexin V/ethidium homodimer III staining assay. The results indicated that compound 4 induced apoptosis more than erlotinib, as shown in Figure 3. The percentages of A549 cells undergoing apoptosis exposed to compound 4 and erlotinib were found to be 56.30% and 53.60%, respectively.

Molecular Docking Studies
Molecular docking studies were carried out for compounds 4 and 1 to designate their binding profiles in the ATP binding site of EGFR compared to erlotinib (Figure 4a). The respective docking scores of compounds 4 and 1 were detected as −7.412 and −7.391 kcal/mol, which were similar to that of erlotinib (−8.895 kcal/mol). This outcome put emphasis on their high binding efficacy to the ATP binding site of EGFR. However, these compounds presented more different binding profiles than erlotinib. Compounds 4 and 1 formed π-cation interactions with Lys721 through its 4-bromophenyl and phenyl substitutions at the second position of 2,4-dihydroindeno[1,2-c]pyrazole, respectively, whereas they missed the important hydrogen bonding with Cys773 and Met769, which erlotinib established in the ATP binding site of EGFR (Figure 4b). This finding could enlighten the weaker EGFR inhibitory potency of compound 4 compared to erlotinib. of A549 cells undergoing apoptosis exposed to compound 4 and erlotinib were found to be 56.30% and 53.60%, respectively. The percentage of apoptotic (green), necrotic or late apoptotic (both green and red), and necrotic (red) cells was quantified by analyzing 100 randomly chosen stained cells in each experiment. All descriptive data were expressed as the Mean ± SD. All experiments were repeated three times. Several crucial pharmacokinetic properties of compounds 1-7 were in silico ascertained. The QPlogBB and predicted central nervous system (CNS) activity values of compounds 1-7 were found within the specified ranges ( Table 2), indicating that compounds 1-7 are able to cross the blood-brain barrier (BBB) and could be effective in CNS metastases in lung cancer. All synthesized compounds also exhibited a high percentage of human oral absorption, between 94.085% and 100%. These compounds violated only one parameter of Jorgensen's rule of three. Besides, they also violated one parameter of Lipinski's rule of five, apart from compounds 4 and 6 because both of them violated two parameters. Overall, these compounds were found to be orally bioavailable drug-like molecules for future studies. kcal/mol, which were similar to that of erlotinib (−8.895 kcal/mol). This outcome put emphasis on their high binding efficacy to the ATP binding site of EGFR. However, these compounds presented more different binding profiles than erlotinib. Compounds 4 and 1 formed π-cation interactions with Lys721 through its 4-bromophenyl and phenyl substitutions at the second position of 2,4-dihydroindeno[1,2-c]pyrazole, respectively, whereas they missed the important hydrogen bonding with Cys773 and Met769, which erlotinib established in the ATP binding site of EGFR (Figure 4b). This finding could enlighten the weaker EGFR inhibitory potency of compound 4 compared to erlotinib.

In Silico Absorption, Distribution, Metabolism, and Excretion (ADME) Studies
Several crucial pharmacokinetic properties of compounds 1-7 were in silico asc tained. The QPlogBB and predicted central nervous system (CNS) activity values of co pounds 1-7 were found within the specified ranges (Table 2), indicating that compoun 1-7 are able to cross the blood-brain barrier (BBB) and could be effective in CNS metas ses in lung cancer. All synthesized compounds also exhibited a high percentage of hum oral absorption, between 94.085% and 100%. These compounds violated only one para eter of Jorgensen's rule of three. Besides, they also violated one parameter of Lipinsk rule of five, apart from compounds 4 and 6 because both of them violated two paramete Overall, these compounds were found to be orally bioavailable drug-like molecules future studies.

Chemistry
All chemicals purchased from commercial suppliers were used without further purification. Melting points (M.p.) were detected on an Electrothermal IA9200 melting point device (Staffordshire, UK) and are uncorrected. IR spectra were recorded on an IRPrestige-21 Fourier Transform IR spectrophotometer (Shimadzu, Tokyo, Japan). NMR ( 1 H and 13 C) analyses were performed on an NMR spectrometer (Bruker, Billerica, MA, USA). HRMS spectra were recorded on a LCMS-IT-TOF system (Shimadzu, Kyoto, Japan). Thin-Layer Chromatography (TLC) was applied to monitor the progress of each chemical reaction and check the purity of each derivative.
General Procedure for the Synthesis of 6-Chloro-7-methoxy-2-aryl-3-[4-(piperidin-1yl)phenyl]-2,4-dihydroindeno[1,2-c]pyrazoles (1-7) A mixture of 5-chloro-6-methoxy-2-[4-(piperidin-1-yl)benzylidene]-2,3-dihydro-1Hinden-1-one (1 mmol) [31], arylhydrazine hydrochloride (3 mmol), and a few drops of acetic acid in ethanol (10 mL) was heated up to 190 • C within 15 min and kept at 190 • C for 20 min under MW irradiation in a reaction vial G30, sealed with a silicone septum and a snap cap with magnetic stirring at 600 rpm in an Anton Paar Monowave 400 Microwave Synthesis Reactor (Graz, Austria), equipped with a ruby thermometer. After the completion of the reaction, the temperature was decreased to 55 • C in the reactor. Then, the reaction mixture was further cooled to room temperature and the precipitate was collected by filtration. The dried product was purified. In experiments, K562 cells and PBMCs were cultured in 24-well and 96-well plates (Asahi Glass Co., Chiba, Japan) at 4 × 10 4 and 1 × 10 6 cells/mL concentrations, respectively, for 48 h. A549 cells were plated (24-well plate) at 1 × 10 4 cells/mL and incubated for 72 h (the optimal cell number was determined in our previous studies) [34][35][36]. The stock solutions of the compounds and erlotinib in concentrations ranging from 1 µM to 10 mM were prepared in DMSO (Wako Pure Chemical Industries, Osaka, Japan) and further diluted with fresh culture medium. The final DMSO concentration was adjusted as 1%, which had no effect on the cell viability [37].

Cell Viability Assay
MTT (Dojindo Molecular Technologies, Kumamoto, Japan) was used to evaluate the cytotoxic effects of the compounds and erlotinib on A549 and K562 cells, and PBMCs, based on the previously described procedures in the literature [37,38]. After cells were exposed to various concentrations (0.1-100 µM) of the compounds for 48 and 72 h at 37 • C, MTT solution was applied and cells were incubated for an additional 4 h. Then, supernatants were discarded, and the formazan crystals were solubilized with a 100 µL DMSO addition. The absorbance of the solution was measured using an Infinite M1000 microplate reader (Tecan, Grödig, Austria) at a wavelength of 570 and 630 nm. All experiments were conducted three times and IC 50 values were specified as the drug concentrations, decreasing absorbance to 50% of control values [39].

Kinase Inhibitory Activity
Manufacturer s instructions (Promega Corporation, Madison, WI, USA) were applied for the kinase profiling assay protocol (TK-1 and TK-2) with small changes [40][41][42]. Ac-cording to this protocol, the kinase stocks in the kinase strips and their substrate stocks in the substrate strips were diluted with 95 µL of 2.5× kinase buffer and 15 µL of 100 µM ATP solutions, respectively. The kinase working stocks (2 µL), 2 µL of the ATP/substrate working stocks, and 1 µL of the tested compound solution (3-100 µM) and erlotinib solution (0.01-100 µM) or 5% DMSO solution in the 384-well plate were used for the kinase reactions. Following the 4 h of incubation at room temperature, the activity of kinases was screened by means of the ADP-Glo Kinase Assay (Promega Corporation, Madison, WI, USA) based on the manufacturer s protocol. The Infinite M1000 microplate reader (Tecan, Grödig, Austria) was used to measure the kinase inhibitory effects of the compounds in a dose-response manner. Besides, the IC 50 values of the tested compounds were determined using ImageJ software.

Detection of Cell Death
A549 cells (1 × 10 4 cells/well) were incubated in each well of a 24-well plate with the most potent compounds in this series at IC 50 concentrations for 15 h. Then, an apoptotic/necrotic cell detection kit (PromoKine, Heidelberg, Germany) was used according to manufacturer s instructions with some modifications [43,44]. Briefly, the cells were washed twice with 1× binding buffer, a staining solution containing 50 µL of 1× binding buffer, 5 µL of FITC-Annexin V solution, and 5 µL of ethidium homodimer III solution, and treated for 30 min at room temperature in a protected-light environment. After washing of cells in 1× binding buffer, cells were analyzed under a Biorevo BZ-9000 all-in-one fluorescence microscope (Keyence, Osaka, Japan). The number of apoptotic cells, late apoptotic or necrotic cells, and necrotic cells was quantified as previously described [45].

Molecular Docking
The X-ray crystallographic structure of EGFR (supplied from the PDB server (PDB code: 4HJO) [46]) was optimized in the protein preparation module. After that, compound 4, compound 1, and erlotinib were optimized with energy minimization in the ligand preparation module. Molecular docking simulations were generated for optimized EGFR and ligands with Grid Generation and Glide/XP protocols (Schrödinger Release 2016-2: Schrödinger, LLC, New York, NY, USA).

Conclusions
In this paper, a MW-assisted technique was employed to efficiently synthesize a new series of 2,4-dihydroindeno[1,2-c]pyrazoles. The MTT assay was conducted to determine their potency as anti-NSCLC agents. Compounds 1, 4, and 7 were found to be more potent than erlotinib on A549 cells. These derivatives were also evaluated for their cytotoxic features on K562 CML cells and PBMCs to assess their selectivity. Based on the SI values of compounds 1, 4, and 7, their anticancer activities were selective. The EGFR TK inhibitory effects of compounds 1, 4, and 7 were also investigated. Among them, p-bromo-substituted compound 4 was the most potent EGFR TKI with an IC 50 value of 17.58 µM. In order to gain a better insight into its kinase selectivity profile, compound 4 was also tested for its inhibitory effects on HER2, HER4, IGF1R, InsR, KDR, PDGFR-α, PDGFR-β, ABL1, BRK, BTK, CSK, FYN A, LCK, LYN B, and SRC. Compound 4 showed more potent inhibitory effects on HER2 and LCK enzymes than erlotinib. According to in vitro kinase profiling assays, it can be concluded that the kinase selectivity profiles of compound 4 and erlotinib are different. Moreover, the annexin V/ethidium homodimer III staining method was applied to determine the effects of compound 4 and erlotinib on apoptosis. Compound 4 induced apoptosis more than erlotinib. Taking into account all in vitro data, compound 4 exerts selective and potent anti-NSCLC activity through the inhibition of EGFR TK, together with the induction of apoptosis, which may counter resistance to chemotherapy and radiotherapy. Molecular docking studies revealed that compound 4 displayed high binding affinity to the ATP binding site of EGFR, similar to erlotinib, but with a more distinct interaction profile. In silico ADME data of compound 4 pointed out its potential as a potential orally bioavailable anti-NSCLC agent endowed with favorable drug-like features. This work, comprising of a combination of biochemical and computational approaches, could represent a rational guideline for further structural modifications of 2,4dihydroindeno[1,2-c]pyrazoles to generate a new class of EGFR TKIs for NSCLC therapy.
Supplementary Materials: The following are available online, Figure S1: The IR spectrum of compound 4; Figure S2: The 1 H NMR spectrum of compound 4; Figure S3: The 13 C NMR spectrum of compound 4; Figure S4: